Norleucine, an analog of the amino acid methionine, can be misincorporated into proteins in place of methionine residues. When expressed in Escherichia coli (E. coli), many heterologous proteins have norleucine mistakenly incorporated in places where methionine residues should appear. The misincorporation of norleucine in proteins, in particular in heterologous proteins produced by recombinant means, is generally considered undesirable due, in part, to the resulting production of altered proteins having undesirable characteristics.
Misincorporation of norleucine at methionine positions during recombinant protein production in E. coli has been observed for over 50 years. (See, e.g., Munier and Cohen (1959) Biochim Biophys Acta 31:378-391; Cohen and Munier (1956) Biochim Biophys Acta 21:592-593; Cohen and Munier (1959) Biochim Biophys Acta 31:347-356; and Cowie et al., (1959) Biochim Biophys Acta 34:39-46.) For example, approximately 14% of methionine residues in methionyl bovine somatotropin (MBS) exhibited norleucine misincorporation during recombinant production of this protein in E coli, and approximately 6% of the methionine residues in native E. coli proteins were also substituted with norleucine. (See Bogosian et al., (1989) J Biol Chem 264:531-9.) In another example, production of interleukin-2 in a minimal medium E. coli fermentation resulted in approximately 19% of the methionine residues in the recombinant protein were substituted with norleucine. (See Tsai et al., (1988) Biochem Biophys Res Commun 156:733-739.) Other studies showed that norleucine residue misincorporation into protein can occur both at internal methionine residues and at the amino terminal methionine residue. (See Brown (1973) Biochim Biophys Acta 294:527-529; and Barker and Bruton (1979) J Mol Biol 133:217-231.)
Norleucine competes with methionine for incorporation into proteins due to the promiscuous nature of the enzyme methionyl tRNA synthetase (MetG). (See Trupin et al., (1966) Biochem Biophys Res Commun 24:50-55; and Fersht and Dingwall (1979) Biochemistry 18:1250-1256.) Kinetic studies with E. coli MetG enzyme showed that acylation of MetG is approximately 4-fold higher with methionine compared to that with norleucine. (See van Hest et al., (2000) Am Chem Soc 122:1282-1288.) Due to the relaxed substrate specificity of MetG, norleucine can substitute for methionine in the acylation reaction, resulting in misincorporation of norleucine into proteins in place of methionine.
Misincorporation of norleucine residues for methionine residues in recombinant protein production is generally considered undesirable. Recombinant proteins or polypeptides containing misincorporated norleucine residues may exhibit altered structural and functional features, such as, for example, altered sensitivity to proteolysis, diminished biological activity, or increased immunogenicity.
Various strategies have been developed to reduce or prevent norleucine misincorporation during recombinant protein production. For example, supplementing cell culture medium with methionine during the fermentation process (by continuous or bolus feed/addition of methionine) has been used to ensure that excess methionine is available to the cells, thus reducing the probability of an incorrect charging of the methionyl tRNA with norleucine. (See, e.g., U.S. Pat. No. 5,599,690.) While continuous or bolus feed/addition of methionine reduced the extent of norleucine misincorporation in recombinant proteins, the operational complexity and cost of the fermentation process may increase. Furthermore, continuous or bolus feed/addition of methionine during fermentation may lead to undesirable dilution of the fermentor contents, resulting in lower cell densities and lower product yields.
Deleting genes involved in the norleucine biosynthetic pathway such as, for example, deleting genes of the leucine operon (leuA, leuB, leuC and leuD) or deleting transaminase encoding genes such as ilvE or tyrB, has also been used to reduce norleucine misincorporation in proteins (See Bogosian et al., (1989) J Biol Chem 264:531-539; Tsai et al., (1989) Biochem Biophys Res Commun 156:733-739; and Randhawa et al., (1994) Biochemistry 33:4352-4362.) The deletion of biosynthetic pathway genes to prevent norleucine misincorporation, however, may require addition of other amino acids (such as leucine or isoleucine) to the culture medium during fermentation as many genes involved in norleucine biosynthesis are also involved in biosynthesis of branched chain amino acids. (See Bogosian et al., (1989) J Biol Chem 264:531-539, see FIG. 8 of the instant specification.)
Another strategy used to prevent norleucine misincorporation involved co-expression of enzymes which degrade norleucine, including, for example, amino acid dehydrogenases and amino acid oxidases. This approach, however, required overexpression of these enzymes, which may not be desirable during recombinant protein production, and may lead to lower recombinant protein yields. (See e.g., United States Patent Application Publication No. US2007/0009995.) In addition, over expression of these enzymes may result in degradation of other analogous amino acids during the fermentation process. Altering the primary amino acid sequence of the polypeptide to be expressed by substituting methionine codons with other codons was also performed to prevent norleucine misincorporation. (See e.g., U.S. Pat. No. 5,698,418.) Such substitutions, however, may lead to diminished activity or structural changes in the resulting protein, a highly undesirable outcome for recombinant protein production in the biotechnology industry.
As noted above, current methods used to prevent norleucine misincorporation during recombinant protein production in microorganisms are associated with various disadvantages; therefore, a need exists for novel methods useful for preventing or reducing norleucine misincorporation in to proteins, in particular during recombinant protein production in microorganisms, such as E. coli. 
The present invention meets this need by providing engineered microorganism host cells effective at preventing norleucine misincorporation during recombinant protein production in microorganisms, such as, for example, bacteria. The present invention provides, inter alia, E. coli host cells comprising mutated metA and metK alleles (i.e., altered metA and metK nucleic acid sequences) which result in methionine production by the microorganism to a degree or extent sufficient to reduce or prevent norleucine misincorporation into proteins and polypeptides. Analysis of recombinant proteins produced utilizing such host cells showed that misincorporation of norleucine residues in place of methionine residues was eliminated. The present invention further demonstrates that fermentation process performance using such E. coli host cells, including growth of the host cells and recombinant protein product titers utilizing such E. coli host cells, was comparable to that observed in control host cells.